氯通道ClC-3、ClC-4和ClC-5在成骨细胞分化中的作用
|
摘要
研究背景
在人的一生中骨总是在不断的改建与重塑,正畸治疗正是利用骨吸收与骨改建的原理治疗错牙合畸形的一门学科,因此对于骨形成与骨改建机理的揭示对于正畸治疗理论基础研究是至关重要的。成骨细胞是参与骨形成及骨改建过程的重要细胞,成骨细胞的增殖、分化及调控是骨形成机理研究的重要内容。研究表明许多因素参与调节细胞的成骨分化,已发现有许多离子通道与细胞的成骨分化有关,如Ca~(2+)、K~+等,作为体内最主要的阴离子通道——氯通道在成骨分化中的作用还知之甚少。
近年来,随着对氯通道结构的深入认识,和一些与氯通道基因突变相关的人类遗传性疾病的发现与研究,氯通道在生物体内的重要作用越来越引起了人们的关注。ClC型氯通道是一大类电压门控型的氯通道,构成了一个大的基因家族。其中,ClC-3、ClC-4和ClC-5为内体系统的ClC型氯通道,由于其基因序列的高度相似性组成了ClC型氯通道家族的一个分支,主要参与神经细胞突触囊泡和细胞内体的酸化作用。近年来,研究发现内体系统的ClC型氯通道基因敲除的小鼠表现出生长阻滞及骨发育的异常,提示我们ClC-3、ClC-4和ClC-5可能与细胞的成骨分化有关。
研究目的
本研究目的在于观察ClC-3、ClC-4和ClC-5氯通道基因在成骨细胞分化过程中的表达变化,从而进一步探求ClC-3、ClC-4和ClC-5在成骨细胞分化中的作用,并初步研究分析其作用机制。
研究方法
体外分离、培养并鉴定小鼠骨髓间充质干细胞,原代培养小鼠颅顶骨来源的成骨细胞及小鼠成骨前体细胞系MC3T3-E1,利用RT-PCR方法检测ClC-3、ClC-4和ClC-5氯通道基因在以上三种成骨谱系细胞中的表达。利用成骨条件培养液对MC3T3-E1细胞进行成骨诱导培养,用RT-PCR及real-time PCR方法检测细胞成骨诱导过程中ClC-3、ClC-4和ClC-5氯通道基因的表达变化。利用脂质体转染含ClC-3、ClC-4和ClC-5氯通道基因全长的质粒,构建瞬时过表达ClC-3、ClC-4和ClC-5氯通道基因的MC3T3-E1细胞体系。同样,利用脂质体转染ClC-3、ClC-4和ClC-5氯通道基因的干扰siRNA,构建干扰ClC-3、ClC-4和ClC-5氯通道基因表达的MC3T3-E1细胞体系。用RT-PCR及real-time PCR方法检测两种基因表达体系中,成骨相关基因的表达变化。在基因过表达体系中,检测细胞体外矿化能力的变化,并检测细胞内细胞器的酸碱变化。用细胞免疫荧光方法,利用激光共聚焦显微镜对MC3T3-E1细胞中的氯通道及TGF-β1进行定位。分析前期研究结果,进一步对氯通道在成骨分化中的作用机制进行研究。
研究结果
RT-PCR结果显示,在小鼠骨髓间充质干细胞、原代培养小鼠颅顶成骨细胞以及小鼠成骨前体细胞系MC3T3-E1细胞中均有ClC-3、ClC-4和ClC-5氯通道基因的阳性表达。在MC3T3-E1细胞成骨诱导培养中,伴随成骨相关基因ALP、BSP、OC、Runx2的表达增强,ClC-3、ClC-4和ClC-5氯通道基因的表达也显著增强。在过表达ClC-3、ClC-4和ClC-5氯通道基因的MC3T3-E1细胞中,成骨相关基因ALP、BSP、OC、Runx2的表达增强,细胞内内体系统的酸化作用加强,细胞体外的矿化能力明显加强。在干扰了ClC-3、ClC-4和ClC-5氯通道基因表达的MC3T3-E1细胞中,成骨相关基因ALP、BSP、OC、Runx2的表达显著降低。在细胞免疫荧光实验中发现,ClC-3氯通道定位于细胞内核周围内体和溶酶体等细胞器。ClC-3与TGF-β1的表达有共定位,且过表达ClC-3基因能明显下调TGF-β1的表达。在过表达ClC-3基因的MC3T3-E1细胞中,干扰Runx2的基因表达后,成骨相关基因ALP、BSP、OC、Runx2、Osx的表达被明显抑制。
结论
ClC-3、ClC-4和ClC-5氯通道存在于小鼠骨髓间充质干细胞、原代培养小鼠颅顶成骨细胞以及小鼠成骨前体细胞系MC3T3-E1等成骨谱系细胞中。ClC-3、ClC-4和ClC-5氯通道对成骨相关基因表达有调节作用,并能增强细胞内细胞器的酸化和细胞体外矿化能力。其中,ClC-3的促成骨分化作用是通过与TGF-β1相关的Runx2途径介导的。ClC-3、ClC-4和ClC-5氯通道基因可作为参与骨形成与骨改建的新基因,有促进成骨分化的作用。
Background
Bone continues remodeling throughout the life. Bone formatioan and remodeling are the basic foundation of orthodontic treatment. The mechanisms of bone resorption and formation are the most important issue of orthodontics researches. Osteoblast is the predominant cell to anticipate the bone formation and remodeling. There are so many factors which influence on the osteogenic differentiation.
The family of voltage gated chloride channels (ClCs) has nine known members in mammals. The ClC chloride channels control the excitability, transepithelial transport, ionic homeostasis, endocytotic trafficking and acidification of vesicles. Basing on their homology similarity, ClCs can be grouped into three branches. The first branch encodes plasma membrane members including ClC-1 and ClC-2. The branch of ClC-3, ClC-4 and ClC-5 are localized in intracellular vesicular system. The third branch including ClC-6 and ClC-7, resides predominantly in intracellular membranes.
The second branch has drawn more attention during recent years due to their contribution to some genetic diseases. Clcn3 gene knockout mice exhibit a complex phenotype including poor growth and kyphosis. The mutations of CLCN5 cause Dent’s disease and Clcn5 gene knockout mice display the identical symptoms of Dent’s disease, such as hyperphosphaturia, hypercalciuria and kidney stones. Clcn5 gene knockout mice also show the abnormal spine and backward growth of teeth. The relationship between ClC-3, ClC-4, and ClC-5 and bone development has been explored recently. ClC-3 contributes to osteoclastic bone resorption in vitro through organelle acidification. ClC-4 and ClC-5 show Cl--H+ antiport activity, like the bacterial homolog ClC-ec1. ClC-5 regulates tooth development through TGF-β1 signal pathway. In this context, we hypothesized that endosomal ClCs might be involved in osteogenic differentiation.
Objective
We conducted the following experiments to examine the expression of Clcn3, Clcn4 and Clcn5 in osteogenic cells and to find out the possible functions of ClC-3, ClC-4 and ClC-5 in osteogenic differentiation.
Methods
Here we used mouse bone marrow stromal stem cells, MC3T3-E1 osteoprogenitor cell line and primarily cultured mouse osteoblasts and detected the expression of Clcn3, Clcn4 and Clcn5 in these cells with RT-PCR. We analyzed the relationships between three endosomal ClCs and the osteogenic phenotype using osteoinductive treatment, overexpressing of ClCs and RNAi of ClCs. We used Alizarin red S staining and intracellular pH staining to explore the biological changes of MC3T3-E1 cells after overexpression of endosomal ClCs. Then, we used RNAi, Western blot and immunofluorescence analysis to explore the molecular mechanisms of ClCs-related function in osteogenic differentiation.
Results
We have detected the positive expression of Clcn3, Clcn4 and Clcn5 in these cells. We found the increased osteogenic markers were in parallel to the increased mRNA levels of ClC-3, ClC-4 and ClC-5 with osteoinductive treatment and overexpressed ClCs. Overexpressed ClCs also promoted the mineralization of MC3T3-E1 cells in vitro and enhanced the acidification of endosomes of cells. Whilst RNAi mediated gene silencing of ClC-3, ClC-4 and ClC-5 down regulated the expression of the four osteogenic markers. Moreover, overexpressed CLC-3 protein co-localized with TGF-β1 in intracellular organelles and downregulated the expression of TGF-β1. Nevertheless, knockdown of Runx2 expression antagonized the effects of ClC-3 in osteodifferentiation and expression of osteogenic markers.
Conclusions
ClC-3, ClC-4 and ClC-5, the subfamily of endosomal ClCs, are expressed in MC3T3-E1 osteoprogenitor cells,primary mouse osteoblasts and mouse bone marrow stem cells. ClC-3, ClC-4 and ClC-5 and some osteogenic marker genes have a positive relationship during osteodifferentiation of MC3T3-E1 cells. ClC-3, ClC-4 and ClC-5 promote the mineralization of MC3T3-E1 cells and take part in endosomal acidification. Furthermore, Runx2 siRNA blocked the function of ClC-3 in the upregulation of gene expression in osteodifferentiation. Further investigation will elucidate the underlying mechanisms and potential use of ClC-3, ClC-4 and ClC-5 as therapeutic targets for treatment of osteogenesis-related diseases.
在人的一生中骨总是在不断的改建与重塑,正畸治疗正是利用骨吸收与骨改建的原理治疗错牙合畸形的一门学科,因此对于骨形成与骨改建机理的揭示对于正畸治疗理论基础研究是至关重要的。成骨细胞是参与骨形成及骨改建过程的重要细胞,成骨细胞的增殖、分化及调控是骨形成机理研究的重要内容。研究表明许多因素参与调节细胞的成骨分化,已发现有许多离子通道与细胞的成骨分化有关,如Ca~(2+)、K~+等,作为体内最主要的阴离子通道——氯通道在成骨分化中的作用还知之甚少。
近年来,随着对氯通道结构的深入认识,和一些与氯通道基因突变相关的人类遗传性疾病的发现与研究,氯通道在生物体内的重要作用越来越引起了人们的关注。ClC型氯通道是一大类电压门控型的氯通道,构成了一个大的基因家族。其中,ClC-3、ClC-4和ClC-5为内体系统的ClC型氯通道,由于其基因序列的高度相似性组成了ClC型氯通道家族的一个分支,主要参与神经细胞突触囊泡和细胞内体的酸化作用。近年来,研究发现内体系统的ClC型氯通道基因敲除的小鼠表现出生长阻滞及骨发育的异常,提示我们ClC-3、ClC-4和ClC-5可能与细胞的成骨分化有关。
研究目的
本研究目的在于观察ClC-3、ClC-4和ClC-5氯通道基因在成骨细胞分化过程中的表达变化,从而进一步探求ClC-3、ClC-4和ClC-5在成骨细胞分化中的作用,并初步研究分析其作用机制。
研究方法
体外分离、培养并鉴定小鼠骨髓间充质干细胞,原代培养小鼠颅顶骨来源的成骨细胞及小鼠成骨前体细胞系MC3T3-E1,利用RT-PCR方法检测ClC-3、ClC-4和ClC-5氯通道基因在以上三种成骨谱系细胞中的表达。利用成骨条件培养液对MC3T3-E1细胞进行成骨诱导培养,用RT-PCR及real-time PCR方法检测细胞成骨诱导过程中ClC-3、ClC-4和ClC-5氯通道基因的表达变化。利用脂质体转染含ClC-3、ClC-4和ClC-5氯通道基因全长的质粒,构建瞬时过表达ClC-3、ClC-4和ClC-5氯通道基因的MC3T3-E1细胞体系。同样,利用脂质体转染ClC-3、ClC-4和ClC-5氯通道基因的干扰siRNA,构建干扰ClC-3、ClC-4和ClC-5氯通道基因表达的MC3T3-E1细胞体系。用RT-PCR及real-time PCR方法检测两种基因表达体系中,成骨相关基因的表达变化。在基因过表达体系中,检测细胞体外矿化能力的变化,并检测细胞内细胞器的酸碱变化。用细胞免疫荧光方法,利用激光共聚焦显微镜对MC3T3-E1细胞中的氯通道及TGF-β1进行定位。分析前期研究结果,进一步对氯通道在成骨分化中的作用机制进行研究。
研究结果
RT-PCR结果显示,在小鼠骨髓间充质干细胞、原代培养小鼠颅顶成骨细胞以及小鼠成骨前体细胞系MC3T3-E1细胞中均有ClC-3、ClC-4和ClC-5氯通道基因的阳性表达。在MC3T3-E1细胞成骨诱导培养中,伴随成骨相关基因ALP、BSP、OC、Runx2的表达增强,ClC-3、ClC-4和ClC-5氯通道基因的表达也显著增强。在过表达ClC-3、ClC-4和ClC-5氯通道基因的MC3T3-E1细胞中,成骨相关基因ALP、BSP、OC、Runx2的表达增强,细胞内内体系统的酸化作用加强,细胞体外的矿化能力明显加强。在干扰了ClC-3、ClC-4和ClC-5氯通道基因表达的MC3T3-E1细胞中,成骨相关基因ALP、BSP、OC、Runx2的表达显著降低。在细胞免疫荧光实验中发现,ClC-3氯通道定位于细胞内核周围内体和溶酶体等细胞器。ClC-3与TGF-β1的表达有共定位,且过表达ClC-3基因能明显下调TGF-β1的表达。在过表达ClC-3基因的MC3T3-E1细胞中,干扰Runx2的基因表达后,成骨相关基因ALP、BSP、OC、Runx2、Osx的表达被明显抑制。
结论
ClC-3、ClC-4和ClC-5氯通道存在于小鼠骨髓间充质干细胞、原代培养小鼠颅顶成骨细胞以及小鼠成骨前体细胞系MC3T3-E1等成骨谱系细胞中。ClC-3、ClC-4和ClC-5氯通道对成骨相关基因表达有调节作用,并能增强细胞内细胞器的酸化和细胞体外矿化能力。其中,ClC-3的促成骨分化作用是通过与TGF-β1相关的Runx2途径介导的。ClC-3、ClC-4和ClC-5氯通道基因可作为参与骨形成与骨改建的新基因,有促进成骨分化的作用。
Background
Bone continues remodeling throughout the life. Bone formatioan and remodeling are the basic foundation of orthodontic treatment. The mechanisms of bone resorption and formation are the most important issue of orthodontics researches. Osteoblast is the predominant cell to anticipate the bone formation and remodeling. There are so many factors which influence on the osteogenic differentiation.
The family of voltage gated chloride channels (ClCs) has nine known members in mammals. The ClC chloride channels control the excitability, transepithelial transport, ionic homeostasis, endocytotic trafficking and acidification of vesicles. Basing on their homology similarity, ClCs can be grouped into three branches. The first branch encodes plasma membrane members including ClC-1 and ClC-2. The branch of ClC-3, ClC-4 and ClC-5 are localized in intracellular vesicular system. The third branch including ClC-6 and ClC-7, resides predominantly in intracellular membranes.
The second branch has drawn more attention during recent years due to their contribution to some genetic diseases. Clcn3 gene knockout mice exhibit a complex phenotype including poor growth and kyphosis. The mutations of CLCN5 cause Dent’s disease and Clcn5 gene knockout mice display the identical symptoms of Dent’s disease, such as hyperphosphaturia, hypercalciuria and kidney stones. Clcn5 gene knockout mice also show the abnormal spine and backward growth of teeth. The relationship between ClC-3, ClC-4, and ClC-5 and bone development has been explored recently. ClC-3 contributes to osteoclastic bone resorption in vitro through organelle acidification. ClC-4 and ClC-5 show Cl--H+ antiport activity, like the bacterial homolog ClC-ec1. ClC-5 regulates tooth development through TGF-β1 signal pathway. In this context, we hypothesized that endosomal ClCs might be involved in osteogenic differentiation.
Objective
We conducted the following experiments to examine the expression of Clcn3, Clcn4 and Clcn5 in osteogenic cells and to find out the possible functions of ClC-3, ClC-4 and ClC-5 in osteogenic differentiation.
Methods
Here we used mouse bone marrow stromal stem cells, MC3T3-E1 osteoprogenitor cell line and primarily cultured mouse osteoblasts and detected the expression of Clcn3, Clcn4 and Clcn5 in these cells with RT-PCR. We analyzed the relationships between three endosomal ClCs and the osteogenic phenotype using osteoinductive treatment, overexpressing of ClCs and RNAi of ClCs. We used Alizarin red S staining and intracellular pH staining to explore the biological changes of MC3T3-E1 cells after overexpression of endosomal ClCs. Then, we used RNAi, Western blot and immunofluorescence analysis to explore the molecular mechanisms of ClCs-related function in osteogenic differentiation.
Results
We have detected the positive expression of Clcn3, Clcn4 and Clcn5 in these cells. We found the increased osteogenic markers were in parallel to the increased mRNA levels of ClC-3, ClC-4 and ClC-5 with osteoinductive treatment and overexpressed ClCs. Overexpressed ClCs also promoted the mineralization of MC3T3-E1 cells in vitro and enhanced the acidification of endosomes of cells. Whilst RNAi mediated gene silencing of ClC-3, ClC-4 and ClC-5 down regulated the expression of the four osteogenic markers. Moreover, overexpressed CLC-3 protein co-localized with TGF-β1 in intracellular organelles and downregulated the expression of TGF-β1. Nevertheless, knockdown of Runx2 expression antagonized the effects of ClC-3 in osteodifferentiation and expression of osteogenic markers.
Conclusions
ClC-3, ClC-4 and ClC-5, the subfamily of endosomal ClCs, are expressed in MC3T3-E1 osteoprogenitor cells,primary mouse osteoblasts and mouse bone marrow stem cells. ClC-3, ClC-4 and ClC-5 and some osteogenic marker genes have a positive relationship during osteodifferentiation of MC3T3-E1 cells. ClC-3, ClC-4 and ClC-5 promote the mineralization of MC3T3-E1 cells and take part in endosomal acidification. Furthermore, Runx2 siRNA blocked the function of ClC-3 in the upregulation of gene expression in osteodifferentiation. Further investigation will elucidate the underlying mechanisms and potential use of ClC-3, ClC-4 and ClC-5 as therapeutic targets for treatment of osteogenesis-related diseases.
引文
[1] Wray D. Structure and function of ion channels. Eur Biophys J 2009;38: 271-2.
[2] Jentsch TJ, Friedrich T, Schriever A, Yamada H. The CLC chloride channel family. Pflugers Arch 1999;437: 783-95.
[3] White MM, Miller C. A voltage-gated anion channel from the electric organ of Torpedo californica. J Biol Chem 1979;254: 10161-6.
[4] Jentsch TJ, Steinmeyer K, Schwarz G. Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature 1990;348: 510-4.
[5] Schmidt-Rose T, Jentsch TJ. Transmembrane topology of a CLC chloride channel. Proc Natl Acad Sci U S A 1997;94: 7633-8.
[6] Ludewig U, Pusch M, Jentsch TJ. Two physically distinct pores in the dimeric ClC-0 chloride channel. Nature 1996;383: 340-3.
[7] Saviane C, Conti F, Pusch M. The muscle chloride channel ClC-1 has a double-barreled appearance that is differentially affected in dominant and recessive myotonia. J Gen Physiol 1999;113: 457-68.
[8] Kubisch C, Schmidt-Rose T, Fontaine B, Bretag AH, Jentsch TJ. ClC-1 chloride channel mutations in myotonia congenita: variable penetrance of mutations shifting the voltage dependence. Hum Mol Genet 1998;7: 1753-60.
[9] Huber S, Braun G, Schroppel B, Horster M. Chloride channels ClC-2 and ICln mRNA expression differs in renal epithelial ontogeny. Kidney Int Suppl 1998;67: S149-51.
[10] Huber SM, Duranton C, Henke G, Van De Sand C, Heussler V, Shumilina E, Sandu CD, Tanneur V, Brand V, Kasinathan RS, Lang KS, Kremsner PG, Hubner CA, Rust MB, Dedek K, Jentsch TJ, Lang F. Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte. J Biol Chem 2004;279: 41444-52.
[11] Roman RM, Smith RL, Feranchak AP, Clayton GH, Doctor RB, Fitz JG. ClC-2 chloride channels contribute to HTC cell volume homeostasis. Am J Physiol Gastrointest Liver Physiol 2001;280: G344-53.
[12] Embark HM, Bohmer C, Palmada M, Rajamanickam J, Wyatt AW, Wallisch S,Capasso G, Waldegger P, Seyberth HW, Waldegger S, Lang F. Regulation of CLC-Ka/barttin by the ubiquitin ligase Nedd4-2 and the serum- and glucocorticoid-dependent kinases. Kidney Int 2004;66: 1918-25.
[13] Uchida S. Physiological role of CLC-K1 chloride channel in the kidney. Nephrol Dial Transplant 2000;15 Suppl 6: 14-5.
[14] Miyamura N, Matsumoto K, Taguchi T, Tokunaga H, Nishikawa T, Nishida K, Toyonaga T, Sakakida M, Araki E. Atypical Bartter syndrome with sensorineural deafness with G47R mutation of the beta-subunit for ClC-Ka and ClC-Kb chloride channels, barttin. J Clin Endocrinol Metab 2003;88: 781-6.
[15] Duan D, Winter C, Cowley S, Hume JR, Horowitz B. Molecular identification of a volume-regulated chloride channel. Nature 1997;390: 417-21.
[16] Friedrich T, Breiderhoff T, Jentsch TJ. Mutational analysis demonstrates that ClC-4 and ClC-5 directly mediate plasma membrane currents. J Biol Chem 1999;274: 896-902.
[17] Steinmeyer K, Schwappach B, Bens M, Vandewalle A, Jentsch TJ. Cloning and functional expression of rat CLC-5, a chloride channel related to kidney disease. J Biol Chem 1995;270: 31172-7.
[18] Gunther W, Luchow A, Cluzeaud F, Vandewalle A, Jentsch TJ. ClC-5, the chloride channel mutated in Dent's disease, colocalizes with the proton pump in endocytotically active kidney cells. Proc Natl Acad Sci U S A 1998;95: 8075-80.
[19] Brandt S, Jentsch TJ. ClC-6 and ClC-7 are two novel broadly expressed members of the CLC chloride channel family. FEBS Lett 1995;377: 15-20.
[20] Kornak U, Kasper D, Bosl MR, Kaiser E, Schweizer M, Schulz A, Friedrich W, Delling G, Jentsch TJ. Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 2001;104: 205-15.
[21] Kasper D, Planells-Cases R, Fuhrmann JC, Scheel O, Zeitz O, Ruether K, Schmitt A, Poet M, Steinfeld R, Schweizer M, Kornak U, Jentsch TJ. Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J 2005;24: 1079-91.
[22] Lange PF, Wartosch L, Jentsch TJ, Fuhrmann JC. ClC-7 requires Ostm1 as a beta-subunit to support bone resorption and lysosomal function. Nature 2006;440:220-3.
[23] Schmieder S, Lindenthal S, Ehrenfeld J. Tissue-specific N-glycosylation of the ClC-3 chloride channel. Biochem Biophys Res Commun 2001;286: 635-40.
[24] Kawasaki M, Suzuki M, Uchida S, Sasaki S, Marumo F. Stable and functional expression of the CIC-3 chloride channel in somatic cell lines. Neuron 1995;14: 1285-91.
[25] Stobrawa SM, Breiderhoff T, Takamori S, Engel D, Schweizer M, Zdebik AA, Bosl MR, Ruether K, Jahn H, Draguhn A, Jahn R, Jentsch TJ. Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. Neuron 2001;29: 185-96.
[26] Yoshikawa M, Uchida S, Ezaki J, Rai T, Hayama A, Kobayashi K, Kida Y, Noda M, Koike M, Uchiyama Y, Marumo F, Kominami E, Sasaki S. CLC-3 deficiency leads to phenotypes similar to human neuronal ceroid lipofuscinosis. Genes Cells 2002;7: 597-605.
[27] Okamoto F, Kajiya H, Toh K, Uchida S, Yoshikawa M, Sasaki S, Kido MA, Tanaka T, Okabe K. Intracellular ClC-3 chloride channels promote bone resorption in vitro through organelle acidification in mouse osteoclasts. Am J Physiol Cell Physiol 2008;294: C693-701.
[28]陈临溪.关永源.氯通道ClC-3研究进展.中国药理学通报2002;5: 1-4.
[29] Wang L, Chen L, Jacob TJ. The role of ClC-3 in volume-activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition. J Physiol 2000;524 Pt 1: 63-75.
[30] Duan D, Zhong J, Hermoso M, Satterwhite CM, Rossow CF, Hatton WJ, Yamboliev I, Horowitz B, Hume JR. Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody. J Physiol 2001;531: 437-44.
[31] Mohammad-Panah R, Ackerley C, Rommens J, Choudhury M, Wang Y, Bear CE. The chloride channel ClC-4 co-localizes with cystic fibrosis transmembrane conductance regulator and may mediate chloride flux across the apical membrane of intestinal epithelia. J Biol Chem 2002;277: 566-74.
[32] Mohammad-Panah R, Harrison R, Dhani S, Ackerley C, Huan LJ, Wang Y, Bear CE.The chloride channel ClC-4 contributes to endosomal acidification and trafficking. J Biol Chem 2003;278: 29267-77.
[33] Picollo A, Pusch M. Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature 2005;436: 420-3.
[34] Mohammad-Panah R, Wellhauser L, Steinberg BE, Wang Y, Huan LJ, Liu XD, Bear CE. An essential role for ClC-4 in transferrin receptor function revealed in studies of fibroblasts derived from Clcn4-null mice. J Cell Sci 2009;122: 1229-37.
[35] Brown D, Stow JL. Protein trafficking and polarity in kidney epithelium: from cell biology to physiology. Physiol Rev 1996;76: 245-97.
[36] Wang SS, Devuyst O, Courtoy PJ, Wang XT, Wang H, Wang Y, Thakker RV, Guggino S, Guggino WB. Mice lacking renal chloride channel, CLC-5, are a model for Dent's disease, a nephrolithiasis disorder associated with defective receptor-mediated endocytosis. Hum Mol Genet 2000;9: 2937-45.
[37] Gunther W, Piwon N, Jentsch TJ. The ClC-5 chloride channel knock-out mouse - an animal model for Dent's disease. Pflugers Arch 2003;445: 456-62.
[38]侯晋.段小红.司徒镇强.氯离子通道ClC-5在大鼠牙胚发育过程中的表达.华西口腔医学杂志2007;25: 444-446.
[39] Duan X, Mao Y, Yang T, Wen X, Wang H, Hou J, Xue Y, Zhang R. ClC-5 regulates dentin development through TGF-beta1 pathway. Arch Oral Biol 2009;54: 1118-24.
[40] Oliveira JM, Sousa RA, Kotobuki N, Tadokoro M, Hirose M, Mano JF, Reis RL, Ohgushi H. The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles. Biomaterials 2009;30: 804-13.
[41] Katagiri T, Takahashi N. Regulatory mechanisms of osteoblast and osteoclast differentiation. Oral Dis 2002;8: 147-59.
[42] Panteghini M, Pagani F. Reference intervals for two bone-derived enzyme activities in serum: bone isoenzyme of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TR-ACP). Clin Chem 1989;35: 180-1.
[43] Havill LM, Hale LG, Newman DE, Witte SM, Mahaney MC. Bone ALP and OC reference standards in adult baboons (Papio hamadryas) by sex and age. J Med Primatol 2006;35: 97-105.
[44] Wada S, Fukawa T, Kamiya S. [Osteocalcin and bone]. Clin Calcium 2007;17: 1673-7.
[45] Leung K, Rajkovic IA, Peters E, Markus I, Van Wyk JJ, Ho KK. Insulin-like growth factor I and insulin down-regulate growth hormone (GH) receptors in rat osteoblasts: evidence for a peripheral feedback loop regulating GH action. Endocrinology 1996;137: 2694-702.
[46] Zanello LP, Norman AW. Stimulation by 1alpha,25(OH)2-vitamin D3 of whole cell chloride currents in osteoblastic ROS 17/2.8 cells. A structure-function study. J Biol Chem 1997;272: 22617-22.
[47] Dallas SL. Measuring interactions between ECM and TGF beta-like proteins. Methods Mol Biol 2000;139: 231-43.
[48] Komori T. Regulation of osteoblast differentiation by transcription factors. J Cell Biochem 2006;99: 1233-9.
[49] Hofer EL, La Russa V, Honegger AE, Bullorsky EO, Bordenave RH, Chasseing NA. Alteration on the expression of IL-1, PDGF, TGF-beta, EGF, and FGF receptors and c-Fos and c-Myc proteins in bone marrow mesenchymal stroma cells from advanced untreated lung and breast cancer patients. Stem Cells Dev 2005;14: 587-94.
[50] Song SJ, Hutmacher D, Nurcombe V, Cool SM. Temporal expression of proteoglycans in the rat limb during bone healing. Gene 2006;379: 92-100.
[51] Naganawa T, Xiao L, Abogunde E, Sobue T, Kalajzic I, Sabbieti M, Agas D, Hurley MM. In vivo and in vitro comparison of the effects of FGF-2 null and haplo-insufficiency on bone formation in mice. Biochem Biophys Res Commun 2006;339: 490-8.
[52] Zhao G, Monier-Faugere MC, Langub MC, Geng Z, Nakayama T, Pike JW, Chernausek SD, Rosen CJ, Donahue LR, Malluche HH, Fagin JA, Clemens TL. Targeted overexpression of insulin-like growth factor I to osteoblasts of transgenic mice: increased trabecular bone volume without increased osteoblast proliferation. Endocrinology 2000;141: 2674-82.
[53] Ghosh-Choudhury N, Windle JJ, Koop BA, Harris MA, Guerrero DL, Wozney JM, Mundy GR, Harris SE. Immortalized murine osteoblasts derived from BMP 2-T-antigen expressing transgenic mice. Endocrinology 1996;137: 331-9.
[54] Chien HH, Lin WL, Cho MI. Down-regulation of osteoblastic cell differentiation by epidermal growth factor receptor. Calcif Tissue Int 2000;67: 141-50.
[55] Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997;89: 765-71.
[56] Doecke JD, Day CJ, Stephens AS, Carter SL, van Daal A, Kotowicz MA, Nicholson GC, Morrison NA. Association of functionally different RUNX2 P2 promoter alleles with BMD. J Bone Miner Res 2006;21: 265-73.
[57]王国红.李祺福. Cbfa1/Runx2与成骨细胞分化调控.生命科学2005;17: 40-44.
[58] Ito Y, Zhang YW. A RUNX2/PEBP2alphaA/CBFA1 mutation in cleidocranial dysplasia revealing the link between the gene and Smad. J Bone Miner Metab 2001;19: 188-94.
[59] Huang L, Teng XY, Cheng YY, Lee KM, Kumta SM. Expression of preosteoblast markers and Cbfa-1 and Osterix gene transcripts in stromal tumour cells of giant cell tumour of bone. Bone 2004;34: 393-401.
[60] Komori T. A fundamental transcription factor for bone and cartilage. Biochem Biophys Res Commun 2000;276: 813-6.
[61] Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002;108: 17-29.
[62] Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev 2002;16: 2813-28.
[63] Phillips JE, Gersbach CA, Wojtowicz AM, Garcia AJ. Glucocorticoid-induced osteogenesis is negatively regulated by Runx2/Cbfa1 serine phosphorylation. J Cell Sci 2006;119: 581-91.
[64] Ding J, Ghali O, Lencel P, Broux O, Chauveau C, Devedjian JC, Hardouin P, Magne D. TNF-alpha and IL-1beta inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells.Life Sci 2009;84: 499-504.
[65] Yoshida K, Oida H, Kobayashi T, Maruyama T, Tanaka M, Katayama T, Yamaguchi K, Segi E, Tsuboyama T, Matsushita M, Ito K, Ito Y, Sugimoto Y, Ushikubi F, Ohuchida S, Kondo K, Nakamura T, Narumiya S. Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation. Proc Natl Acad Sci U S A 2002;99: 4580-5.
[66] McCarthy TL, Chang WZ, Liu Y, Centrella M. Runx2 integrates estrogen activity in osteoblasts. J Biol Chem 2003;278: 43121-9.
[67] Lambertini E, Penolazzi L, Tavanti E, Schincaglia GP, Zennaro M, Gambari R, Piva R. Human estrogen receptor alpha gene is a target of Runx2 transcription factor in osteoblasts. Exp Cell Res 2007;313: 1548-60.
[68] Wiper-Bergeron N, St-Louis C, Lee JM. CCAAT/Enhancer binding protein beta abrogates retinoic acid-induced osteoblast differentiation via repression of Runx2 transcription. Mol Endocrinol 2007;21: 2124-35.
[69] Leontiadis LJ, Papakonstantinou MP, Georgoussi Z. Regulator of G protein signaling 4 confers selectivity to specific G proteins to modulate mu- and delta-opioid receptor signaling. Cell Signal 2009;21: 1218-28.
[70] Meldolesi J. Calcium signalling. Oscillation, activation, expression. Nature 1998;392: 863, 865-6.
[71] Zahanich I, Graf EM, Heubach JF, Hempel U, Boxberger S, Ravens U. Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells. J Bone Miner Res 2005;20: 1637-46.
[72] van der Eerden BC, Hoenderop JG, de Vries TJ, Schoenmaker T, Buurman CJ, Uitterlinden AG, Pols HA, Bindels RJ, van Leeuwen JP. The epithelial Ca2+ channel TRPV5 is essential for proper osteoclastic bone resorption. Proc Natl Acad Sci U S A 2005;102: 17507-12.
[73] Masuyama R, Vriens J, Voets T, Karashima Y, Owsianik G, Vennekens R, Lieben L, Torrekens S, Moermans K, Vanden Bosch A, Bouillon R, Nilius B, Carmeliet G. TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts. Cell Metab 2008;8: 257-65.
[74] Zanello LP, Norman AW. Multiple molecular mechanisms of 1 alpha,25(OH)2-vitamin D3 rapid modulation of three ion channel activities in osteoblasts. Bone 2003;33: 71-9.
[75] Zanello LP, Norman A. 1alpha,25(OH)2 vitamin D3 actions on ion channels in osteoblasts. Steroids 2006;71: 291-7.
[76] Okabe K, Okamoto F, Kajiya H, Takada K, Soeda H. Estrogen directly acts on osteoclasts via inhibition of inward rectifier K+ channels. Naunyn Schmiedebergs Arch Pharmacol 2000;361: 610-20.
[77] Ypey DL, Weidema AF, Hold KM, Van der Laarse A, Ravesloot JH, Van Der Plas A, Nijweide PJ. Voltage, calcium, and stretch activated ionic channels and intracellular calcium in bone cells. J Bone Miner Res 1992;7 Suppl 2: S377-87.
[78] Yellowley CE, Hancox JC, Skerry TM, Levi AJ. Whole-cell membrane currents from human osteoblast-like cells. Calcif Tissue Int 1998;62: 122-32.
[79] Ravesloot JH, Ypey DL, Nijweide PJ, Buisman HP, Vriejheid-Lammers T. Three voltage-activated K+ conductances and an ATP-activated conductance in freshly isolated embryonic chick osteoclasts. Pflugers Arch 1989;414 Suppl 1: S166-7.
[80] Maki M, Miyazaki H, Nakajima K, Yamane J, Niisato N, Morihara T, Kubo T, Marunaka Y. Chloride-dependent acceleration of cell cycle via modulation of Rb and cdc2 in osteoblastic cells. Biochem Biophys Res Commun 2007;361: 1038-43.
[81] Yang JY, Jung JY, Cho SW, Choi HJ, Kim SW, Kim SY, Kim HJ, Jang CH, Lee MG, Han J, Shin CS. Chloride intracellular channel 1 regulates osteoblast differentiation. Bone 2009;45: 1175-85.
[82] Yamamoto-Mizuma S, Wang GX, Hume JR. P2Y purinergic receptor regulation of CFTR chloride channels in mouse cardiac myocytes. J Physiol 2004;556: 727-37.
[83] Coelho MJ, Fernandes MH. Human bone cell cultures in biocompatibility testing. Part II: effect of ascorbic acid, beta-glycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials 2000;21: 1095-102.
[84] Choi KM, Seo YK, Yoon HH, Song KY, Kwon SY, Lee HS, Park JK. Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation. J Biosci Bioeng 2008;105: 586-94.
[85] Banerjee C, McCabe LR, Choi JY, Hiebert SW, Stein JL, Stein GS, Lian JB. Runthomology domain proteins in osteoblast differentiation: AML3/CBFA1 is a major component of a bone-specific complex. J Cell Biochem 1997;66: 1-8.
[86] Carmichael GG. Medicine: silencing viruses with RNA. Nature 2002;418: 379-80.
[87] Tang YB, Liu YJ, Zhou JG, Wang GL, Qiu QY, Guan YY. Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G/S phase arrest in rat basilar arterial smooth muscle cells. Cell Prolif 2008;41: 775-85.
[88] Yin Z, Tong Y, Zhu H, Watsky MA. ClC-3 is required for LPA-activated Cl- current activity and fibroblast-to-myofibroblast differentiation. Am J Physiol Cell Physiol 2008;294: C535-42.
[89] Cheng G, Shao Z, Chaudhari B, Agrawal DK. Involvement of chloride channels in TGF-beta1-induced apoptosis of human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2007;293: L1339-47.
[90] Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res 1992;7: 683-92.
[91] Jentsch TJ. Chloride and the endosomal-lysosomal pathway: emerging roles of CLC chloride transporters. J Physiol 2007;578: 633-40.
[92] Piwon N, Gunther W, Schwake M, Bosl MR, Jentsch TJ. ClC-5 Cl- -channel disruption impairs endocytosis in a mouse model for Dent's disease. Nature 2000;408: 369-73.
[93] Grabe M, Oster G. Regulation of organelle acidity. J Gen Physiol 2001;117: 329-44.
[94] Nabavi N, Urukova Y, Cardelli M, Aubin JE, Harrison RE. Lysosome dispersion in osteoblasts accommodates enhanced collagen production during differentiation. J Biol Chem 2008;283: 19678-90.
[95] Hara-Chikuma M, Yang B, Sonawane ND, Sasaki S, Uchida S, Verkman AS. ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation. J Biol Chem 2005;280: 1241-7.
[96] Li X, Wang T, Zhao Z, Weinman SA. The ClC-3 chloride channel promotes acidification of lysosomes in CHO-K1 and Huh-7 cells. Am J Physiol Cell Physiol 2002;282: C1483-91.
[97] Lyons RM, Keski-Oja J, Moses HL. Proteolytic activation of latent transforminggrowth factor-beta from fibroblast-conditioned medium. J Cell Biol 1988;106: 1659-65.
[98] Massague J. Receptors for the TGF-beta family. Cell 1992;69: 1067-70.
[99] Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 1997;390: 465-71.
[100] Perlman R, Schiemann WP, Brooks MW, Lodish HF, Weinberg RA. TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation. Nat Cell Biol 2001;3: 708-14.
[101] Liu W, Toyosawa S, Furuichi T, Kanatani N, Yoshida C, Liu Y, Himeno M, Narai S, Yamaguchi A, Komori T. Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. J Cell Biol 2001;155: 157-66.
[102] Tu Q, Valverde P, Chen J. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells. Biochem Biophys Res Commun 2006;341: 1257-65.
[2] Jentsch TJ, Friedrich T, Schriever A, Yamada H. The CLC chloride channel family. Pflugers Arch 1999;437: 783-95.
[3] White MM, Miller C. A voltage-gated anion channel from the electric organ of Torpedo californica. J Biol Chem 1979;254: 10161-6.
[4] Jentsch TJ, Steinmeyer K, Schwarz G. Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature 1990;348: 510-4.
[5] Schmidt-Rose T, Jentsch TJ. Transmembrane topology of a CLC chloride channel. Proc Natl Acad Sci U S A 1997;94: 7633-8.
[6] Ludewig U, Pusch M, Jentsch TJ. Two physically distinct pores in the dimeric ClC-0 chloride channel. Nature 1996;383: 340-3.
[7] Saviane C, Conti F, Pusch M. The muscle chloride channel ClC-1 has a double-barreled appearance that is differentially affected in dominant and recessive myotonia. J Gen Physiol 1999;113: 457-68.
[8] Kubisch C, Schmidt-Rose T, Fontaine B, Bretag AH, Jentsch TJ. ClC-1 chloride channel mutations in myotonia congenita: variable penetrance of mutations shifting the voltage dependence. Hum Mol Genet 1998;7: 1753-60.
[9] Huber S, Braun G, Schroppel B, Horster M. Chloride channels ClC-2 and ICln mRNA expression differs in renal epithelial ontogeny. Kidney Int Suppl 1998;67: S149-51.
[10] Huber SM, Duranton C, Henke G, Van De Sand C, Heussler V, Shumilina E, Sandu CD, Tanneur V, Brand V, Kasinathan RS, Lang KS, Kremsner PG, Hubner CA, Rust MB, Dedek K, Jentsch TJ, Lang F. Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte. J Biol Chem 2004;279: 41444-52.
[11] Roman RM, Smith RL, Feranchak AP, Clayton GH, Doctor RB, Fitz JG. ClC-2 chloride channels contribute to HTC cell volume homeostasis. Am J Physiol Gastrointest Liver Physiol 2001;280: G344-53.
[12] Embark HM, Bohmer C, Palmada M, Rajamanickam J, Wyatt AW, Wallisch S,Capasso G, Waldegger P, Seyberth HW, Waldegger S, Lang F. Regulation of CLC-Ka/barttin by the ubiquitin ligase Nedd4-2 and the serum- and glucocorticoid-dependent kinases. Kidney Int 2004;66: 1918-25.
[13] Uchida S. Physiological role of CLC-K1 chloride channel in the kidney. Nephrol Dial Transplant 2000;15 Suppl 6: 14-5.
[14] Miyamura N, Matsumoto K, Taguchi T, Tokunaga H, Nishikawa T, Nishida K, Toyonaga T, Sakakida M, Araki E. Atypical Bartter syndrome with sensorineural deafness with G47R mutation of the beta-subunit for ClC-Ka and ClC-Kb chloride channels, barttin. J Clin Endocrinol Metab 2003;88: 781-6.
[15] Duan D, Winter C, Cowley S, Hume JR, Horowitz B. Molecular identification of a volume-regulated chloride channel. Nature 1997;390: 417-21.
[16] Friedrich T, Breiderhoff T, Jentsch TJ. Mutational analysis demonstrates that ClC-4 and ClC-5 directly mediate plasma membrane currents. J Biol Chem 1999;274: 896-902.
[17] Steinmeyer K, Schwappach B, Bens M, Vandewalle A, Jentsch TJ. Cloning and functional expression of rat CLC-5, a chloride channel related to kidney disease. J Biol Chem 1995;270: 31172-7.
[18] Gunther W, Luchow A, Cluzeaud F, Vandewalle A, Jentsch TJ. ClC-5, the chloride channel mutated in Dent's disease, colocalizes with the proton pump in endocytotically active kidney cells. Proc Natl Acad Sci U S A 1998;95: 8075-80.
[19] Brandt S, Jentsch TJ. ClC-6 and ClC-7 are two novel broadly expressed members of the CLC chloride channel family. FEBS Lett 1995;377: 15-20.
[20] Kornak U, Kasper D, Bosl MR, Kaiser E, Schweizer M, Schulz A, Friedrich W, Delling G, Jentsch TJ. Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 2001;104: 205-15.
[21] Kasper D, Planells-Cases R, Fuhrmann JC, Scheel O, Zeitz O, Ruether K, Schmitt A, Poet M, Steinfeld R, Schweizer M, Kornak U, Jentsch TJ. Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J 2005;24: 1079-91.
[22] Lange PF, Wartosch L, Jentsch TJ, Fuhrmann JC. ClC-7 requires Ostm1 as a beta-subunit to support bone resorption and lysosomal function. Nature 2006;440:220-3.
[23] Schmieder S, Lindenthal S, Ehrenfeld J. Tissue-specific N-glycosylation of the ClC-3 chloride channel. Biochem Biophys Res Commun 2001;286: 635-40.
[24] Kawasaki M, Suzuki M, Uchida S, Sasaki S, Marumo F. Stable and functional expression of the CIC-3 chloride channel in somatic cell lines. Neuron 1995;14: 1285-91.
[25] Stobrawa SM, Breiderhoff T, Takamori S, Engel D, Schweizer M, Zdebik AA, Bosl MR, Ruether K, Jahn H, Draguhn A, Jahn R, Jentsch TJ. Disruption of ClC-3, a chloride channel expressed on synaptic vesicles, leads to a loss of the hippocampus. Neuron 2001;29: 185-96.
[26] Yoshikawa M, Uchida S, Ezaki J, Rai T, Hayama A, Kobayashi K, Kida Y, Noda M, Koike M, Uchiyama Y, Marumo F, Kominami E, Sasaki S. CLC-3 deficiency leads to phenotypes similar to human neuronal ceroid lipofuscinosis. Genes Cells 2002;7: 597-605.
[27] Okamoto F, Kajiya H, Toh K, Uchida S, Yoshikawa M, Sasaki S, Kido MA, Tanaka T, Okabe K. Intracellular ClC-3 chloride channels promote bone resorption in vitro through organelle acidification in mouse osteoclasts. Am J Physiol Cell Physiol 2008;294: C693-701.
[28]陈临溪.关永源.氯通道ClC-3研究进展.中国药理学通报2002;5: 1-4.
[29] Wang L, Chen L, Jacob TJ. The role of ClC-3 in volume-activated chloride currents and volume regulation in bovine epithelial cells demonstrated by antisense inhibition. J Physiol 2000;524 Pt 1: 63-75.
[30] Duan D, Zhong J, Hermoso M, Satterwhite CM, Rossow CF, Hatton WJ, Yamboliev I, Horowitz B, Hume JR. Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody. J Physiol 2001;531: 437-44.
[31] Mohammad-Panah R, Ackerley C, Rommens J, Choudhury M, Wang Y, Bear CE. The chloride channel ClC-4 co-localizes with cystic fibrosis transmembrane conductance regulator and may mediate chloride flux across the apical membrane of intestinal epithelia. J Biol Chem 2002;277: 566-74.
[32] Mohammad-Panah R, Harrison R, Dhani S, Ackerley C, Huan LJ, Wang Y, Bear CE.The chloride channel ClC-4 contributes to endosomal acidification and trafficking. J Biol Chem 2003;278: 29267-77.
[33] Picollo A, Pusch M. Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature 2005;436: 420-3.
[34] Mohammad-Panah R, Wellhauser L, Steinberg BE, Wang Y, Huan LJ, Liu XD, Bear CE. An essential role for ClC-4 in transferrin receptor function revealed in studies of fibroblasts derived from Clcn4-null mice. J Cell Sci 2009;122: 1229-37.
[35] Brown D, Stow JL. Protein trafficking and polarity in kidney epithelium: from cell biology to physiology. Physiol Rev 1996;76: 245-97.
[36] Wang SS, Devuyst O, Courtoy PJ, Wang XT, Wang H, Wang Y, Thakker RV, Guggino S, Guggino WB. Mice lacking renal chloride channel, CLC-5, are a model for Dent's disease, a nephrolithiasis disorder associated with defective receptor-mediated endocytosis. Hum Mol Genet 2000;9: 2937-45.
[37] Gunther W, Piwon N, Jentsch TJ. The ClC-5 chloride channel knock-out mouse - an animal model for Dent's disease. Pflugers Arch 2003;445: 456-62.
[38]侯晋.段小红.司徒镇强.氯离子通道ClC-5在大鼠牙胚发育过程中的表达.华西口腔医学杂志2007;25: 444-446.
[39] Duan X, Mao Y, Yang T, Wen X, Wang H, Hou J, Xue Y, Zhang R. ClC-5 regulates dentin development through TGF-beta1 pathway. Arch Oral Biol 2009;54: 1118-24.
[40] Oliveira JM, Sousa RA, Kotobuki N, Tadokoro M, Hirose M, Mano JF, Reis RL, Ohgushi H. The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles. Biomaterials 2009;30: 804-13.
[41] Katagiri T, Takahashi N. Regulatory mechanisms of osteoblast and osteoclast differentiation. Oral Dis 2002;8: 147-59.
[42] Panteghini M, Pagani F. Reference intervals for two bone-derived enzyme activities in serum: bone isoenzyme of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TR-ACP). Clin Chem 1989;35: 180-1.
[43] Havill LM, Hale LG, Newman DE, Witte SM, Mahaney MC. Bone ALP and OC reference standards in adult baboons (Papio hamadryas) by sex and age. J Med Primatol 2006;35: 97-105.
[44] Wada S, Fukawa T, Kamiya S. [Osteocalcin and bone]. Clin Calcium 2007;17: 1673-7.
[45] Leung K, Rajkovic IA, Peters E, Markus I, Van Wyk JJ, Ho KK. Insulin-like growth factor I and insulin down-regulate growth hormone (GH) receptors in rat osteoblasts: evidence for a peripheral feedback loop regulating GH action. Endocrinology 1996;137: 2694-702.
[46] Zanello LP, Norman AW. Stimulation by 1alpha,25(OH)2-vitamin D3 of whole cell chloride currents in osteoblastic ROS 17/2.8 cells. A structure-function study. J Biol Chem 1997;272: 22617-22.
[47] Dallas SL. Measuring interactions between ECM and TGF beta-like proteins. Methods Mol Biol 2000;139: 231-43.
[48] Komori T. Regulation of osteoblast differentiation by transcription factors. J Cell Biochem 2006;99: 1233-9.
[49] Hofer EL, La Russa V, Honegger AE, Bullorsky EO, Bordenave RH, Chasseing NA. Alteration on the expression of IL-1, PDGF, TGF-beta, EGF, and FGF receptors and c-Fos and c-Myc proteins in bone marrow mesenchymal stroma cells from advanced untreated lung and breast cancer patients. Stem Cells Dev 2005;14: 587-94.
[50] Song SJ, Hutmacher D, Nurcombe V, Cool SM. Temporal expression of proteoglycans in the rat limb during bone healing. Gene 2006;379: 92-100.
[51] Naganawa T, Xiao L, Abogunde E, Sobue T, Kalajzic I, Sabbieti M, Agas D, Hurley MM. In vivo and in vitro comparison of the effects of FGF-2 null and haplo-insufficiency on bone formation in mice. Biochem Biophys Res Commun 2006;339: 490-8.
[52] Zhao G, Monier-Faugere MC, Langub MC, Geng Z, Nakayama T, Pike JW, Chernausek SD, Rosen CJ, Donahue LR, Malluche HH, Fagin JA, Clemens TL. Targeted overexpression of insulin-like growth factor I to osteoblasts of transgenic mice: increased trabecular bone volume without increased osteoblast proliferation. Endocrinology 2000;141: 2674-82.
[53] Ghosh-Choudhury N, Windle JJ, Koop BA, Harris MA, Guerrero DL, Wozney JM, Mundy GR, Harris SE. Immortalized murine osteoblasts derived from BMP 2-T-antigen expressing transgenic mice. Endocrinology 1996;137: 331-9.
[54] Chien HH, Lin WL, Cho MI. Down-regulation of osteoblastic cell differentiation by epidermal growth factor receptor. Calcif Tissue Int 2000;67: 141-50.
[55] Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997;89: 765-71.
[56] Doecke JD, Day CJ, Stephens AS, Carter SL, van Daal A, Kotowicz MA, Nicholson GC, Morrison NA. Association of functionally different RUNX2 P2 promoter alleles with BMD. J Bone Miner Res 2006;21: 265-73.
[57]王国红.李祺福. Cbfa1/Runx2与成骨细胞分化调控.生命科学2005;17: 40-44.
[58] Ito Y, Zhang YW. A RUNX2/PEBP2alphaA/CBFA1 mutation in cleidocranial dysplasia revealing the link between the gene and Smad. J Bone Miner Metab 2001;19: 188-94.
[59] Huang L, Teng XY, Cheng YY, Lee KM, Kumta SM. Expression of preosteoblast markers and Cbfa-1 and Osterix gene transcripts in stromal tumour cells of giant cell tumour of bone. Bone 2004;34: 393-401.
[60] Komori T. A fundamental transcription factor for bone and cartilage. Biochem Biophys Res Commun 2000;276: 813-6.
[61] Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 2002;108: 17-29.
[62] Akiyama H, Chaboissier MC, Martin JF, Schedl A, de Crombrugghe B. The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev 2002;16: 2813-28.
[63] Phillips JE, Gersbach CA, Wojtowicz AM, Garcia AJ. Glucocorticoid-induced osteogenesis is negatively regulated by Runx2/Cbfa1 serine phosphorylation. J Cell Sci 2006;119: 581-91.
[64] Ding J, Ghali O, Lencel P, Broux O, Chauveau C, Devedjian JC, Hardouin P, Magne D. TNF-alpha and IL-1beta inhibit RUNX2 and collagen expression but increase alkaline phosphatase activity and mineralization in human mesenchymal stem cells.Life Sci 2009;84: 499-504.
[65] Yoshida K, Oida H, Kobayashi T, Maruyama T, Tanaka M, Katayama T, Yamaguchi K, Segi E, Tsuboyama T, Matsushita M, Ito K, Ito Y, Sugimoto Y, Ushikubi F, Ohuchida S, Kondo K, Nakamura T, Narumiya S. Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation. Proc Natl Acad Sci U S A 2002;99: 4580-5.
[66] McCarthy TL, Chang WZ, Liu Y, Centrella M. Runx2 integrates estrogen activity in osteoblasts. J Biol Chem 2003;278: 43121-9.
[67] Lambertini E, Penolazzi L, Tavanti E, Schincaglia GP, Zennaro M, Gambari R, Piva R. Human estrogen receptor alpha gene is a target of Runx2 transcription factor in osteoblasts. Exp Cell Res 2007;313: 1548-60.
[68] Wiper-Bergeron N, St-Louis C, Lee JM. CCAAT/Enhancer binding protein beta abrogates retinoic acid-induced osteoblast differentiation via repression of Runx2 transcription. Mol Endocrinol 2007;21: 2124-35.
[69] Leontiadis LJ, Papakonstantinou MP, Georgoussi Z. Regulator of G protein signaling 4 confers selectivity to specific G proteins to modulate mu- and delta-opioid receptor signaling. Cell Signal 2009;21: 1218-28.
[70] Meldolesi J. Calcium signalling. Oscillation, activation, expression. Nature 1998;392: 863, 865-6.
[71] Zahanich I, Graf EM, Heubach JF, Hempel U, Boxberger S, Ravens U. Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells. J Bone Miner Res 2005;20: 1637-46.
[72] van der Eerden BC, Hoenderop JG, de Vries TJ, Schoenmaker T, Buurman CJ, Uitterlinden AG, Pols HA, Bindels RJ, van Leeuwen JP. The epithelial Ca2+ channel TRPV5 is essential for proper osteoclastic bone resorption. Proc Natl Acad Sci U S A 2005;102: 17507-12.
[73] Masuyama R, Vriens J, Voets T, Karashima Y, Owsianik G, Vennekens R, Lieben L, Torrekens S, Moermans K, Vanden Bosch A, Bouillon R, Nilius B, Carmeliet G. TRPV4-mediated calcium influx regulates terminal differentiation of osteoclasts. Cell Metab 2008;8: 257-65.
[74] Zanello LP, Norman AW. Multiple molecular mechanisms of 1 alpha,25(OH)2-vitamin D3 rapid modulation of three ion channel activities in osteoblasts. Bone 2003;33: 71-9.
[75] Zanello LP, Norman A. 1alpha,25(OH)2 vitamin D3 actions on ion channels in osteoblasts. Steroids 2006;71: 291-7.
[76] Okabe K, Okamoto F, Kajiya H, Takada K, Soeda H. Estrogen directly acts on osteoclasts via inhibition of inward rectifier K+ channels. Naunyn Schmiedebergs Arch Pharmacol 2000;361: 610-20.
[77] Ypey DL, Weidema AF, Hold KM, Van der Laarse A, Ravesloot JH, Van Der Plas A, Nijweide PJ. Voltage, calcium, and stretch activated ionic channels and intracellular calcium in bone cells. J Bone Miner Res 1992;7 Suppl 2: S377-87.
[78] Yellowley CE, Hancox JC, Skerry TM, Levi AJ. Whole-cell membrane currents from human osteoblast-like cells. Calcif Tissue Int 1998;62: 122-32.
[79] Ravesloot JH, Ypey DL, Nijweide PJ, Buisman HP, Vriejheid-Lammers T. Three voltage-activated K+ conductances and an ATP-activated conductance in freshly isolated embryonic chick osteoclasts. Pflugers Arch 1989;414 Suppl 1: S166-7.
[80] Maki M, Miyazaki H, Nakajima K, Yamane J, Niisato N, Morihara T, Kubo T, Marunaka Y. Chloride-dependent acceleration of cell cycle via modulation of Rb and cdc2 in osteoblastic cells. Biochem Biophys Res Commun 2007;361: 1038-43.
[81] Yang JY, Jung JY, Cho SW, Choi HJ, Kim SW, Kim SY, Kim HJ, Jang CH, Lee MG, Han J, Shin CS. Chloride intracellular channel 1 regulates osteoblast differentiation. Bone 2009;45: 1175-85.
[82] Yamamoto-Mizuma S, Wang GX, Hume JR. P2Y purinergic receptor regulation of CFTR chloride channels in mouse cardiac myocytes. J Physiol 2004;556: 727-37.
[83] Coelho MJ, Fernandes MH. Human bone cell cultures in biocompatibility testing. Part II: effect of ascorbic acid, beta-glycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials 2000;21: 1095-102.
[84] Choi KM, Seo YK, Yoon HH, Song KY, Kwon SY, Lee HS, Park JK. Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation. J Biosci Bioeng 2008;105: 586-94.
[85] Banerjee C, McCabe LR, Choi JY, Hiebert SW, Stein JL, Stein GS, Lian JB. Runthomology domain proteins in osteoblast differentiation: AML3/CBFA1 is a major component of a bone-specific complex. J Cell Biochem 1997;66: 1-8.
[86] Carmichael GG. Medicine: silencing viruses with RNA. Nature 2002;418: 379-80.
[87] Tang YB, Liu YJ, Zhou JG, Wang GL, Qiu QY, Guan YY. Silence of ClC-3 chloride channel inhibits cell proliferation and the cell cycle via G/S phase arrest in rat basilar arterial smooth muscle cells. Cell Prolif 2008;41: 775-85.
[88] Yin Z, Tong Y, Zhu H, Watsky MA. ClC-3 is required for LPA-activated Cl- current activity and fibroblast-to-myofibroblast differentiation. Am J Physiol Cell Physiol 2008;294: C535-42.
[89] Cheng G, Shao Z, Chaudhari B, Agrawal DK. Involvement of chloride channels in TGF-beta1-induced apoptosis of human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2007;293: L1339-47.
[90] Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res 1992;7: 683-92.
[91] Jentsch TJ. Chloride and the endosomal-lysosomal pathway: emerging roles of CLC chloride transporters. J Physiol 2007;578: 633-40.
[92] Piwon N, Gunther W, Schwake M, Bosl MR, Jentsch TJ. ClC-5 Cl- -channel disruption impairs endocytosis in a mouse model for Dent's disease. Nature 2000;408: 369-73.
[93] Grabe M, Oster G. Regulation of organelle acidity. J Gen Physiol 2001;117: 329-44.
[94] Nabavi N, Urukova Y, Cardelli M, Aubin JE, Harrison RE. Lysosome dispersion in osteoblasts accommodates enhanced collagen production during differentiation. J Biol Chem 2008;283: 19678-90.
[95] Hara-Chikuma M, Yang B, Sonawane ND, Sasaki S, Uchida S, Verkman AS. ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation. J Biol Chem 2005;280: 1241-7.
[96] Li X, Wang T, Zhao Z, Weinman SA. The ClC-3 chloride channel promotes acidification of lysosomes in CHO-K1 and Huh-7 cells. Am J Physiol Cell Physiol 2002;282: C1483-91.
[97] Lyons RM, Keski-Oja J, Moses HL. Proteolytic activation of latent transforminggrowth factor-beta from fibroblast-conditioned medium. J Cell Biol 1988;106: 1659-65.
[98] Massague J. Receptors for the TGF-beta family. Cell 1992;69: 1067-70.
[99] Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 1997;390: 465-71.
[100] Perlman R, Schiemann WP, Brooks MW, Lodish HF, Weinberg RA. TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation. Nat Cell Biol 2001;3: 708-14.
[101] Liu W, Toyosawa S, Furuichi T, Kanatani N, Yoshida C, Liu Y, Himeno M, Narai S, Yamaguchi A, Komori T. Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. J Cell Biol 2001;155: 157-66.
[102] Tu Q, Valverde P, Chen J. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells. Biochem Biophys Res Commun 2006;341: 1257-65.